1. Field of the Invention
The present invention relates to novel hepatocyte-specific tri-valent ligands based on nitrilotriacetic acid (NTA) having three N-acetylgalactosamine (GalNAc) or three or six lactose (Lac) moieties attached to the NTA structure.
2. Related Art
Asialoglycoprotein receptor (ASGPR) of the liver is known to specifically bind to glycopeptides or glycoproteins having Gal or GalNAc on an end. When liver lesion occurs, the number of ASGPR will decrease. Therefore, it is desirable to develop high-affinity ligands for ASGPR with multiple Gal or GalNAc terminal residues useful for imaging of ASGPR activities.
This type of liver receptor activity imaging agents has the potential of quantifying the liver storage function and also has the ability to determine the residual liver function in a patient to allow clinicians to determine whether a liver transplantation is necessary or not. In Taiwan, there are 3,000,000 patients with hepatitis B, 500,000 patients with hepatitis C, and numerous patients with drug-induced hepatitis, all of whom are at high risk of liver failure and need regular assessment of liver storage function.
Presently, the peptides or proteins to be acquired multivalency with saccharide groups known in the art include albumin, tyrosine-glutamyl-glutamic acid (YEE), tyro sine-aspartyl-aspartic acid (YDD), and tyro sine-glutamyl-glutamyl-glutamic acid (YEEE).
Tc-99m-Galactosyl-Serum-Albumin (Tc-99m GSA) is known as a liver receptor imaging agent and has been used clinically in Japan. But, GSA is a carbohydrate-modified protein based on human serum albumin, which is a biological product having a very high molecular weight of about 67 kD. In such a product, it is difficult to know the exact position of modification and the precise number of the sugar attachment, not to mention DTPA (diethylene triamine pentaacetate) further attaches to GSA for labeling. In contrast, YEE, YDD, and YEEE are based on peptides having a molecular weight of only about 1-2 kD, and the exact structures, including the DTPA, are known. Thus the quality control procedures are significantly simplified compared to those procedures used with GSA.
YEE(ahGalNAc)3 and YDD(ahGalNAc)3 were first reported by Lee (1983), but their poor solubility limited their development. YEEE(ahGalNAc)3 is an improved version reported by Chen (TW1240002, 2000). However, the overall yield is still unsatisfactory, the cost of GalNAc is considerably high, and it is not commercially available.
In 1983, Lee et al. reported that the binding affinity of divalent GalNAc-containing peptide by rat hepatocytes is 1000 times stronger than that of its monovalent GalNAc counterpart and the binding affinity between trivalent GalNAc ligand with hepatocytes receptor is 106 times greater than that of monovalent GalNAc ligand peptide with a single chain.
It should be noted that in YEE, YDD, and YEEE derivatives, a branching scaffold is provided by peptides rather than sugars as in natural glycoproteins, for example, γ-glutamyl-glutamic acid (abbreviated as “EE”), and β-aspartyl-aspartic acid (abbreviated as “DD”). Both EE and DD have three COOH functional groups being exposed and can thus be joined with three w-amino glycosides of GalNAc. However, the disadvantage of these compounds and tedious chemical synthetic process impeded their further development.
Therefore, it is desirable to develop a new type of high affinity reagents for hepatocytes (targeting ASGPR) which are convenient to synthesize and processing adequate solubility.